The HER2/neu receptor is overexpressed in approximately one-third of all breast cancers and is associated with a poor clinical outcome. This research proposal will establish the effects of Herceptin (anti-HER2/neu) alone or in combination with chemotherapy drugs on cell proliferation and apoptosis in breast cancer lines expressing HER2/neu. To test this hypothesis, assays that measure cell proliferation, such as DNA content and colony formation assays, and apoptosis, including flow cytometry and DNA condensation experiments, will be used to determine cell outcome in response to Herceptin and chemotherapy drugs. To assess the functionality of signal transduction cascades (Akt, ERK, JNK and p38MAPK), phospho-specific antibodies will be used as a readout for activity. Additionally, selective pharmacological agents will be employed to modulate specific pathways involved in Herceptin and chemotherapy-induced growth inhibition and apoptosis. These studies will ultimately be confirmed with dominant-negative and gain-of function mutant proteins for each signaling pathway being tested. These mutants will be engineered with a cell-permeable TAT sequence to allow transduction into the cell. Also, transient transfection of the mutant proteins will be done. This proposal will establish a model whereby the modulation of signal transduction cascades by clinically important drugs will result in the inhibition of breast cancer cell growth. The results from this proposal will offer a greater understanding of the basis of cancer treatment and provide information for the rational development of new therapies.